Download biomedica product listRat NT-proBNP ELISA | BI-1204R
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Category number
BI-1204R
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Method
Sandwich ELISA, HRPO/TMB, 12×8-well detachable strips
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Sample type
Serum, Plasma
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Sample volume
10 µl / well
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Assay time
2 h / 1 h / 30 min
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Sensitivity
LOD: 21 pg/ml; LLOQ: 50 pg/ml
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Standard range
0 – 3200 pg/ml (0 / 100 / 200 / 400 / 800 / 1600 / 3200)
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Precision
Within-run (n=3): ≤ 6% CV; In-between-run (n=9): ≤ 4% CV
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Regulatory status
Research use only
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Validation Data
See validation data tab for: precision, accuracy, dilution linearity etc.
Rat NT-proBNP ELISA Product Overview
The Rat NT-proBNP immunoassay is a 3.5 hour, 96-well sandwich ELISA for the quantitative determination of rat NT-proBNP in serum or plasma.
Rat NT-proBNP ELISA Assay Principle
The Rat NT-proBNP ELISA kit is a sandwich enzyme immunoassay for the quantitative determination of NT-proBNP in rat samples.
Figure explaining the principle of a sandwich ELISA:
In a first step, ASYBUF (Assay buffer), STD/CTRL/Sample (Standard, Control, Sample) and detection antibody (biotinylated polyclonal anti-rat NT-proBNP antibody, AB) are pipetted into the respective wells of the microtiter strips, which are pre-coated with a polyclonal anti-rat NT-proBNP antibody. Rat NT-proBNP present in the sample binds to the pre-coated antibody in the well and forms a sandwich with the detection antibody.
After a first wash step, which removes non-specifically unbound material, the conjugate (Streptavidin-HRPO) is added and reacts with the detection antibody. After another washing step, the substrate (TMB, tetramethylbenzidine) is pipetted into the wells.
The enzyme catalysed color change of the substrate is directly proportional to the amount of rat NT-proBNP present in the sample. This color change is detectable with a standard microtiter plate ELISA reader. The concentration of rat NT-proBNP in the sample is determined directly from the dose response curve. The kit utilizes recombinant rat NT-proBNP as a calibrator.
Rat NT-proBNP ELISA Typical Standard Curve
The figure below shows a typical standard curve for the rat NT proBNP ELISA. The immunoassay is calibrated against recombinant rat NT-proBNP peptide:
Rat NT-proBNP ELISA Kit Components
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Contents |
Description |
Quantity |
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PLATE |
Detachable microtiter strips pre-coated with polyclonal anti-rat NT-proBNP antibody, packed in an aluminum bag with desiccant |
12 x 8 tests |
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WASHBUF |
Wash buffer concentrate 20x, clear cap |
1 x 50 ml |
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ASYBUF |
Assay buffer, ready-to-use, red cap |
1 x 7 ml |
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STOCK STD |
Stock standard containing recombinant rat NT-proBNP (lyophilized) with concentration 3200 pg/ml, red cap |
1 vial |
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CTRL |
Control, exact concentration on the label (lyophilized), yellow cap |
1 vial |
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CONJ |
Streptavidin-HRPO conjugate, ready to use, amber cap |
1 x 13 ml |
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SUB |
Substrate (TMB solution), blue cap, ready to use |
1 x 13 ml |
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STOP |
Stop solution, white cap, ready to use |
1 x 7 ml |
Serum and plasma are suitable for use in this assay. Do not change sample type during studies. We recommend duplicate measurements for all samples, standards, and control.
Serum & Plasma
Collect venous blood samples in standardized blood collection tubes. Perform serum or plasma separation by centrifugation according to supplier’s instructions of the blood collection devices. The acquired serum or plasma samples should be measured as soon as possible. For longer storage aliquot samples and store at -25°C or lower. Samples are stable for up to five freeze-thaw cycles.
Lipemic or haemolysed samples may give erroneous results. Samples should be mixed well before assaying. We recommend duplicates for all values.
Samples with values above STD7 (3200 pg/ml) can be diluted with ASYBUF (Assay buffer).
Reagent Preparation
Bring all reagents to room temperature before use.
Wash Buffer
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1. |
Bring the WASHBUF concentrate to room temperature. Crystals in the buffer concentrate will dissolve at room temperature. |
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2. |
Dilute the WASHBUF concentrate 1:20, e.g. 50 ml WASHBUF + 950 ml distilled or deionized water. Only use diluted WASHBUF when performing the assay. |
The diluted WASHBUF is stable up to one month at 4°C (2-8°C).
STOCK Standard and Control
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1. |
Pipette 200µl of distilled or deionized water into stock standard (STOCK STD) and control (CTRL) vials. The exact concentration is printed on the label of each vial. |
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2. |
Leave at room temperature (18-26°C) for 15 min. Vortex gently. |
Reconstituted STOCK STD and CTRL are stable at -25°C or lower until expiry date stated on the label. STOCK STD and CTRL are stable up to five freeze-thaw cycles.
Preparation of the standard curve:
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1. |
Use polypropylene tubes and mark them as STD6 to STD1 as shown in below graph. |
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2. |
Mark STOCK STD as STD7. |
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3. |
Pipette 50 µl of ASYBUF (assay buffer) into each tube marked as STD6 to STD1. |
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4. |
Prepare a two-fold serial dilution to obtain STD6 to STD2. |
Graph of standard curve preparation:
Sample Preparation
Bring samples to room temperature and mix samples gently to ensure the samples are homogenous. We recommend duplicate measurements for all samples. Samples for which the optical density (OD) value exceeds the highest point of the standard range (STD7, 3200 pg/ml) can be diluted with ASYBUF.
Rat NT-proBNP ELISA Assay Protocol
Read the entire protocol before beginning the assay.
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1. |
Bring reagents and samples to room temperature (18-26°C). |
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2. |
Mark position for STD/SAMPLE/CTRL (Standard/Sample/Control) on the protocol sheet. |
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3. |
Take microtiter strips out of the aluminum bag. Store unused strips with desiccant at 4°C in the aluminum bag. Strips are stable until the expiry date stated on the label. |
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4. |
Pipette 50 µl ASYBUF (assay buffer, red cap) into each well. |
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5. |
Add 10 µl STD/CTRL/SAMPLE in duplicate into the respective wells. Swirl gently. |
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6. |
Add 50 µl AB (biotinylated anti-rat NT-proBNP, green cap) into each well. Swirl gently. |
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7. |
Cover the plate tightly and incubate for 2 hours at room temperature (18-26°C). |
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8. |
Aspirate and wash wells 5 x with 300 µl diluted WASHBUF. After the final wash, remove remaining WASHBUF by strongly tapping plate against a paper towel. |
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9. |
Add 100 µl CONJ (conjugate, amber cap) into each well. Swirl gently. |
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10. |
Cover the plate tightly and incubate for 1 hour at room temperature (18-26°C). |
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11. |
Aspirate and wash wells 5 x with 300 µl diluted WASHBUF. After the final wash, remove remaining WASHBUF by strongly tapping plate against a paper towel. |
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12. |
Add 100 µl SUB (Substrate) into each well. |
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13. |
Incubate for 30 min at room temperature (18-26°C) in the dark. |
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15. |
Add 50 µl STOP (Stop solution) into each well. Swirl gently. |
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16. |
Measure absorbance immediately at 450 nm with reference 630 nm, if available. |
Calculation of Results
Read the optical density (OD) of all wells on a plate reader using 450 nm wavelength (reference wavelength 630 nm). Construct a standard curve from the absorbance read-outs of the standards using commercially available software capable of generating a four-parameter logistic (4-PL) fit. Alternatively, plot the standards’ concentration on the x-axis against the mean absorbance for each standard on the y-axis and draw a best fit curve through the points on the graph. Curve fitting algorithms other than 4-PL have not been validated and will need to be evaluated by the user. Obtain sample concentrations from the standard curve.
Samples with analyte concentrations outside of the calibration range of the assay (3200 pg/ml) should be diluted with assay buffer.
Concentrations of high-measuring samples that have been diluted during sample preparation must be multiplied by the dilution factor.
The quality control (QC) protocol supplied with the kit shows the results of the final release QC for each kit at production date. Data for OD obtained by customers may differ due to various influences and/or due to the normal decrease of signal intensity during shelf life. However, this does not affect validity of results as long as an OD of 1.0 or more is obtained for STD7 and the value of the CTRL is in range (target range see label).
NT-proBNP Protein
BNP is mainly expressed by the ventricular myocardium in response to volume overload and increased filling pressure. It is synthesized and secreted by cardiomyocytes. Mature BNP consists of 108 amino acids (proBNP or BNP-108). ProBNP is cleaved during secretion in a 1:1 ratio resulting in physiologically active BNP-32 and the biologically inactive 76 amino acid NT-proBNP (http://www.uniprot.org/uniprot/P16860#PRO_0000001532). NT-proBNP (1-76) has greater plasma stability and a much longer biological half-life (90-120 minutes) than BNP, being considered as the preferred laboratory marker.
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Molecular weight |
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Cellular localisation |
Secreted |
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Post-translational modifications |
Cleaved into 1 chains Brain natriuretic peptide 45. |
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Sequence similarities |
- |
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Alternative names |
Natriuretic peptides B, Brain natriuretic factor prohormone, preproBNP, proBNP, NT-proBNP |
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Entrez/NCBI ID |
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Genecards |
NPPB link: https://www.ncbi.nlm.nih.gov/gene/25105 |
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PDB |
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OMIM |
600295 link: https://www.omim.org/entry/600295 |
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Protein Atlas |
- |
| Uniprot ID |
P13205 · ANFB_RAT |
NT-proBNP Function
BNP has a key role in cardiovascular homeostasis with biological actions including natriuresis, diuresis, vasorelaxation, and inhibition of renin and aldosterone secretion. A high concentration of BNP in the bloodstream is indicative of heart failure.
Literature
- Heart Failure Association of the European Society of Cardiology practical guidance on the use of natriuretic peptide concentrations. Mueller C et al., Eur J Heart Fail, 21(6):715-731 (2019).
- Utility of the Amino-Terminal Fragment of Pro Brain Natriuretic Peptide in Plasma. For the Evaluation of Cardiac Dysfunction in elderly Patients in Primary Health Care. Alehagen U et al., Clin Chem, 49:8; 1337-1346 (2003).
- Head-to-head comparison of N-terminal pro-brain natriuretic peptide, brain natriuretic peptide and N-terminal proatrial natriuretic peptide in diagnosing left ventricular dysfunction. Hammerer-Lercher et al., Clin Chim Acta, 310(2),193-197 (2001).
- Molecular Signaling Mechanisms and Function of Natriuretic Peptide Receptor-A in the Pathophysiology of Cardiovascular Homeostasis. Pandey KN et al., Front Physiol, 19;12:693099 (2021).
- NT-ProBNP and mortality across the spectrum of glucose tolerance in the general US population. Ciardullo S et al., Cardiovasc Diabetol, 7;21(1):236 (2022).
- Plasma pro-B-type natriuretic peptide in the general population: screening for left ventricular hypertrophy and systolic dysfunction. Goetze JP et al., Eur Heart J, 27: 3004-3010 (2006).
- N-terminal pro brain natriuretic peptide reference values in community-dwelling older adults. Braisch U et al., ESC Heart Fail, 9(3):1703-1712 (2022).
Calibration
This rat NT-proBNP immunoassay is calibrated against recombinant rat NT-proBNP (1-76) peptide.
Rat NT-proBNP ELISA Detection Limit & Sensitivity
To determine the sensitivity of the rat NT-proBNP ELISA, experiments measuring the lower limit of detection (LOD) and the lower limit of quantification (LLOQ) were conducted.
The LOD, also called the detection limit, is the lowest point at which a signal can be distinguished above the background signal, i.e. the signal that is measured in rat NT-proBNP free sample (five independent measurements) plus three times the standard deviation of the measurements.
The LLOQ, or sensitivity of an assay, is the lowest concentration at which an analyte can be accurately quantified. The criteria for accurate quantification at the LLOQ are an analyte recovery between 75% and 125% and a coefficient of variation (CV) of less than 25%. To determine the LLOQ, standard 2, i.e. the lowest standard containing rat NT-proBNP, is diluted, measured five times and its concentration back calculated. The lowest dilution, which meets both criteria, is reported as the LLOQ.
The following values were determined for the rat NT-proBNP ELISA:
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LOD |
21 pg/ml |
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LLOQ |
50 pg/ml |
Rat NT-proBNP ELISA Precision
The precision of an ELISA is defined as its ability to measure the same concentration consistently within the same experiments carried out by one operator (within-run precision or repeatability) and across several experiments using the same samples but conducted by several operators at different locations using different ELISA lots (in-between-run precision or reproducibility).
Within-Run Precision
Two samples of known concentration were tested three times to assess within-run precision, also called intra-assay precision. The experiment was performed by one operator using a single plate.
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ID |
Within-Run Precision n |
Mean Rat NT-proBNP [pg/ml] |
SD [pg/ml] |
CV [%] |
|
Sample 1 |
3 |
153 |
98 |
6 |
|
Sample 2 |
3 |
1600 |
27 |
2 |
In-Between-Run Precision
Two samples of known concentration were tested nine times to assess in-between-run precision, also called inter-assay precision. The experiment was performed by multiple operators using plates and kit components from different lots.
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ID |
In-Between Run Precision n |
Mean rat NT-proBNP [pg/ml] |
SD [pg/ml] |
CV (%) |
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Sample 1 |
9 |
199 |
8.8 |
4 |
|
Sample 2 |
9 |
1603 |
344.4 |
2 |
Rat NT-proBNP ELISA Accuracy
The accuracy of an ELISA is defined as the precision with which it can recover samples of known concentrations.
The recovery of the rat NT-proBNP ELISA was measured by adding recombinant rat NT-proBNP to rat samples containing a known concentration of endogenous rat NT-proBNP. The %recovery of the spiked concentration was calculated as the percentage of measured compared over the expected value. All our ELISAs are expected to have %recovery rates within 20% of the nominal value of the sample.
This table shows the summary of the recovery experiments in the rat NT-proBNP ELISA in different sample matrices:
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% Recovery |
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Sample Matrix |
n |
+ 1600 pg/ml |
+400 pg/ml |
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Mean |
Range |
Mean |
Range |
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Serum |
6 |
90 |
83 - 97 |
86 |
79 - 95 |
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Plasma |
3 |
88 |
84 - 94 |
81 |
78 - 83 |
Data showing recovery of rat NT-proBNP in rat serum samples:
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Rat NT-proBNP [pg/ml] |
% Recovery |
|||||
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Sample Matrix |
ID |
Reference |
+ 1600 pg/ml |
+ 400 pg/ml |
+ 1600 pg/ml |
+ 400 pg/ml |
|
Serum |
S1 |
161 | 1631 | 521 | 97 |
95 |
|
Serum |
S2 |
800 | 1724 | 1017 | 83 | 79 |
|
Serum |
S3 |
388 | 1707 | 704 | 95 | 91 |
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Serum |
S4 |
289 | 1533 | 572 | 87 | 80 |
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Serum |
S5 |
198 | 1533 | 496 | 90 | 81 |
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Serum |
S6 |
479 | 1663 | 779 | 89 | 90 |
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Mean |
90 | 86 | ||||
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Min |
83 | 70 | ||||
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Max |
97 | 95 | ||||
Data showing recovery of rat NT-proBNP in rat plasma samples:
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Rat NT-proBNP [pg/ml] |
% Recovery |
|||||
|
Sample Matrix |
ID |
Reference |
+ 1600 pg/ml |
+ 400 pg/ml |
+ 1600 pg/ml |
+ 400 pg/ml |
|
Plasma |
P1 |
161 | 1631 | 521 | 97 | 95 |
|
Plasma |
P2 |
118 | 1562 | 417 | 94 | 78 |
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Plasma |
P3 |
78 | 1423 | 400 | 87 |
83 |
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Mean |
88 | 81 | ||||
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Min |
84 | 78 | ||||
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Max |
94 | 83 | ||||
Rat NT-proBNP ELISA Dilution Linearity & Parallelism
Tests of dilution linearity and parallelism ensure that both endogenous and recombinant samples containing rat NT-proBNP behave in a dose dependent manner and are not affected by matrix effects. Dilution linearity assesses the accuracy of measurements in diluted human samples spiked with known concentrations of recombinant analyte. By contrast, parallelism refers to dilution linearity in rat samples and provides evidence that the endogenous analyte behaves the same way as the recombinant one. Dilution linearity and parallelism are assessed for each sample type and are considered acceptable if the results are within 20% of the expected concentration.
Dilution linearity was assessed by serially diluting rat samples spiked with 1600 pg/ml recombinant rat NT-proBNP with assay buffer.
The figure and table below show the mean recovery and range of serially diluted recombinant rat NT-proBNP in rat serum and plasma samples:
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% Recovery of recombinant Rat NT-proBNP in diluted rat samples |
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Sample Matrix |
n |
1+1 |
1+3 |
1+7 |
|||
|
Mean |
Range |
Mean |
Range |
Mean |
Range | ||
|
Serum |
3 |
101 | 99 - 103 | 102 | 99 - 103 | 103 | 93 - 108 |
|
Plasma |
2 |
107 | 98 - 116 | 112 | 103 - 121 | 123 | 112 - 135 |
Data showing dilution linearity of 1600 pg/ml recombinant rat NT-proBNP spiked into serum samples (ref) containing endogenous rat NT-proBNP:
|
Rat NT-proBNP (pg/ml) |
% Recovery |
|||||||
|
Sample Matrix |
ID |
Ref +1600 pg/ml |
1+1 |
1+3 |
1+7 |
1+1 |
1+3 |
1+7 |
|
Serum |
S1 |
1912 | 973 | 494 | 254 | 102 | 103 | 106 |
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Serum |
S2 |
1671 | 825 | 415 | 195 | 99 | 99 | 93 |
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Serum |
S3 |
1793 | 899 | 449 | 235 | 103 | 103 | 108 |
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Mean |
101 |
102 |
103 |
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Data showing dilution linearity of 1600 pg/ml recombinant rat NT-proBNP spiked into plasma samples (ref) containing endogenous rat NT-proBNP:
|
Rat NT-proBNP (pg/ml) |
% Recovery |
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Sample Matrix |
ID |
Ref +1600 pg/ml |
1+1 |
1+3 |
1+7 |
1+1 |
1+3 |
1+7 |
|
Plasma |
P1 |
1441 | 835 | 435 | 243 | 116 | 121 | 135 |
|
Plasma |
P2 |
1562 | 768 | 404 | 218 | 98 | 103 | 112 |
| Mean R[%] |
107 |
112 |
123 |
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Parallelism was assessed by serially diluting samples containing endogenous rat NT-proBNP with assay buffer.
The figure and table below show the mean recovery and range of serially diluted endogenous rat NT-proBNP in serum samples:
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% Recovery of endogenous rat NT-proBNP in diluted rat samples |
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|
Sample Matrix |
n |
1+1 |
1+3 |
1+7 |
|||
|
Mean |
Range |
Mean |
Range |
Mean |
Range | ||
|
Serum |
5 |
106 | 100 - 112 | 116 | 95 - 125 | 113 | 97 - 119 |
Data showing paralellism of endogenous rat NT-proBNP in serum samples:
|
Rat NT-proBNP (pg/ml) |
% Recovery |
|||||||
|
Sample Matrix |
ID |
Ref |
1+1 |
1+3 |
1+7 |
1+1 |
1+3 |
1+7 |
|
Serum |
S1 |
1240 | 665 | 386 | 185 | 107 | 125 | 119 |
|
Serum |
S2 |
838 | 417 | 236 | 120 | 100 | 113 | 115 |
|
Serum |
S3 |
902 | 503 | 278 | 130 | 112 | 123 | 115 |
|
Serum |
S4 |
1119 | 579 | 303 | 136 | 103 | 108 | 97 |
|
Serum |
S5 |
672 | 348 | 186 | 100 | 104 | 111 | 119 |
|
Mean |
105 | 116 | 113 | |||||
|
Min |
100 |
108 |
97 |
|||||
|
Max |
112 |
125 |
119 |
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Rat NT-proBNP ELISA Specificity
The specificity of an ELISA is defined as its ability to exclusively recognize the analyte of interest.
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Rat NT-proBNP (pg/ml) |
% Competition |
|||||
|
Sample Matrix |
ID |
Reference |
Reference + rat NT-proBNP capture antibody |
|||
|
Serum, recombinant |
STD7 |
3200 | 3 | 100 | ||
|
Serum |
S1 |
515 | 0 | 100 | ||
|
Serum |
S2 |
542 | 4 | 99 | ||
|
Serum |
S3 |
340 | 0 | 100 | ||
|
Serum |
S4 |
1120 | 0 | 100 | ||
|
Serum |
S5 |
142 | 0 | 1000 | ||
|
Serum |
S6 |
519 | 0 | 100 | ||
|
Serum |
S7 |
1100 | 0 | 100 | ||
|
Mean |
100 | |||||
Sample Stability
The stability of endogenous rat NT-proBNP was tested by comparing its concentration measured in samples that had undergone 5 freeze-thaw cycles.
Reference samples were freeze-thawed once. The mean recovery of sample concentration after 5 freeze-thaw cycles is 107%.
|
Rat NT-proBNP (OD) |
% Recovery 5 F/T vs ref |
||||
|
Sample ID |
Reference |
1x |
3x |
5x |
|
|
Serum 1 |
0.377 | 0.411 | 0.406 | 0.395 | 105 |
|
Serum2 |
0.277 | 0.282 | 0.288 | 0.298 | 108 |
|
Serum3 |
0.332 | 0.358 | 0.352 | 0.358 | 108 |
|
Plasma1 |
0.703 | 0.730 | 0.731 | 0.759 | 108 |
|
Mean |
107 |
||||
Samples can undergo up to 5 freeze-thaw cycles.
Sample Values
Rat NT-proBNP Values in Apparently Healthy Animals
To provide an estimation of rat NT-proBNP values from apparently healthy rats we measured a small sample number of rat serum/plasma samples deriving from rat strains with different genetic backgrounds. Rat NT-proBNP reference values may differ from other rat strains.
A summary of the results is shown below:
|
Rat NT-proBNP (pg/ml) |
% Detectable |
||||
|
Sample Matrix (n*) |
Mean |
Median |
Minimum |
Maximum |
|
|
Serum (6) |
82 | 74 | 0 | 169 | 83 |
|
Plasma** (14) |
47 | 22 | 0 | 185 | 86 |
*: samples derived from rat strains with different genetic backgrounds. Rat NT-proBNP reference values may differ from other rat strains. , **: EDTA and Citrate plasma
We recommended establishing the normal range for each laboratory.
Rat NT-proBNP Values in a Clinical Cohort
Values of rat NT-proBNP in healthy rats and rats from kidney disease model adriamycin nephropathy (AN) and renal mass reduction (RMR) were measured.
|
ID |
Rat NT-proBNP (pg/ml) |
|
|
Control group |
Adriamycin nephropathy |
|
| #1 | 384 | 788 |
| #2 | 379 | 623 |
| #3 | 270 | 490 |
| #4 | 405 | 688 |
| #5 | 237 | 840 |
|
#6 |
n.a. |
922 |
|
#7 |
n.a. |
723 |
|
#8 |
n.a. |
624 |
n.a. = not available
|
|
Rat NT-proBNP (pg/ml) |
|
|
Control group |
Adriamycin nephropathy |
|
| Sample number | 5 | 8 |
| Minimum | 270 | 490 |
| Median | 379 | 706 |
| Maximum | 405 | 922 |
| Mean | 335 | 712 |
|
ID |
Rat NT-proBNP (pg/ml) |
|
|
Control group |
Renal mass reduction (RMR) |
|
| #1 | 90 | 275 |
| #2 | 129 | 587 |
| #3 | 45 | 797 |
| #4 | 169 | 814 |
| #5 | n.a. | 708 |
|
#6 |
n.a. | 463 |
|
#7 |
n.a. | 1724 |
n.a. = not available
|
|
Rat NT-proBNP (pg/ml) |
|
|
Control group |
Renal mass reduction (RMR) |
|
| Sample number | 4 | 7 |
| Minimum | 45 | 275 |
| Median | 110 | 708 |
| Maximum | 169 | 1724 |
| Mean | 108 | 767 |
Graph: Rat NT-proBNP level changes in heparinized plasma
